Abstract

The oncoproteins P3k (homolog of the catalytic subunit of class IA
phosphoinositide 3-kinase) and Akt (protein kinase B) induce oncogenic
transformation of chicken embryo fibroblasts. The transformed cells
show constitutive phosphorylation of the positive regulator of
translation p70S6 kinase (S6K) and of the eukaryotic initiation factor
4E-BP1 binding protein (4E-BP1), a negative regulator of translation.
Phosphorylation activates S6K and inactivates 4E-BP1. A mutant of Akt
that retains kinase activity but does not induce phosphorylation of S6K
or of 4E-BP1 fails to transform chicken embryo fibroblasts, suggesting
a correlation between the oncogenicity of Akt and phosphorylation of
S6K and 4E-BP1. The macrolide antibiotic rapamycin effectively blocks
oncogenic transformation induced by either P3k or Akt but does not
reduce the transforming activity of 11 other oncoproteins. Rapamycin
inhibits the kinase mTOR, an important regulator of translation, and
this inhibition requires binding of the antibiotic to the immunophilin
FKBP12. Displacement of rapamycin from FKBP12 relieves the inhibition
of mTOR and also restores P3k-induced transformation. These data are in
accord with the hypothesis that transformation by P3k or Akt involves
intervention in translational controls.

The two oncoproteins P3k and
Akt were originally isolated from tumorigenic retroviruses (1, 2). P3k
is the homolog of the catalytic subunit of phosphoinositide (PI)
3-kinase, a lipid kinase that phosphorylates phosphatidylinositol at
the D3 position and affects multiple cellular functions, many related
to growth and differentiation (3–6). Akt (also called PKB) is a
serine–threonine protein kinase; it is a downstream target of PI
3-kinase (7–11). Akt binds to the products of PI 3-kinase,
phosphatidylinositol 3,4-biphosphate and phosphatidylinositol
3,4,5-triphosphate, with its pleckstrin homology domain. It then
becomes activated by phosphorylation at threonine 308 and serine
473 through the action of the 3-phosphoinositide-dependent kinases PDK1
and PDK2 (12, 13).

Akt affects numerous downstream targets either directly or indirectly
(7–11). These can be broadly classified into two groups: (i) survival
and death factors and (ii) proteins controlling translation. Among the
first group are the pro-apoptotic proteins Bad (14, 15) and
caspase 9 (16) and the growth-inhibitory proteins glycogen synthase
kinase-3 beta (17) and the forkhead transcription factors FKHR,
FKHR-L1, and AFX, all of which are down-regulated by Akt (18–20). Also
in this category is the kinase IKK alpha, a positive regulator of
NF-κB, which is up-regulated by Akt (21–23). The second category
consists of the kinase mTOR (mammalian target of rapamycin, other
acronyms: FRAP, RAFT) and its downstream targets p70 S6 kinase (S6K)
and the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, also
called PHAS-1) (24–27). S6K is activated by mTOR-dependent
phosphorylation and controls the translation of 5′TOP mRNAs, so named
for the presence of an oligopyrimidine tract at their 5′ termini (28).
These messages code for ribosomal proteins and elongation factors; the
oligopyrimidine tract mediates coordinate translational regulation in a
growth-dependent fashion. 4E-BP is inactivated by mTOR-dependent
phosphorylation (29–32). Underphosphorylated 4E-BP binds to the
eukaryotic initiation factor 4E (eIF4E, the cap-binding protein) and
prevents it from assembling the translation initiation complex at the
cap of the mRNA. Phosphorylation abolishes this inhibitory function.

Here, we provide evidence that oncogenic transformation by P3k and Akt
is dependent on targets that control translation: transforming activity
is correlated with phosphorylation and activation of S6K and is
eliminated by the mTOR inhibitor rapamycin. The suggested involvement
of translational control in oncogenic transformation is specific for
P3k and Akt; transformation induced by numerous other oncoproteins is
not inhibited by rapamycin.

For serum starvation, cells were cultured in Ham's F-10 medium with
0.5% FCS and 0.1% chicken serum. After 40 h, the medium was
replaced with plain F-10 medium, and the culture was further incubated
for 2 h. The cells were then stimulated with 50 ng/ml PDGF (Life
Technologies, Grand Island, NY). For rapamycin treatment, rapamycin (10
ng/ml) was added to the culture 2 h before the addition of PDGF.

Results

Constitutive Phosphorylation of S6K and 4E-BP1 in CEF Transformed
by P3k and Akt.

S6K and 4E-BP1 are phosphorylated in a rapamycin-sensitive manner,
suggesting a dependence of this process on the mTOR kinase (47–52).
Phosphorylation of S6K on the rapamycin-sensitive threonine 389
correlates well with S6K activation in vivo (53). We
examined the phosphorylation of S6K in Western blots using a
phospho-S6K-specific antibody (Fig.
1A). In serum-starved CEF,
uninfected or infected with the RCAS vector alone, phosphorylation of
S6K was low, but PDGF induced strong phosphorylation of threonine 389
within 15 min. In contrast, CEF transformed by P3k or Akt showed strong
phosphorylation of threonine 389, even under conditions of serum
deprivation, suggesting that S6K is constitutively activated in the
transformed cells. Control Western blots with
phosphorylation-independent antibody against S6K showed that the
expression levels of S6K were not altered in P3k or Akt transformation
or by stimulation with PDGF. We also investigated the phosphorylation
of 4E-BP1 in Western blots using a phospho-specific antibody that
recognizes 4E-BP1 phosphorylated on serine 65 (Fig. 1B).
This site is phosphorylated in response to growth factors in a
rapamycin-sensitive manner (29). Again, in uninfected and RCAS-infected
CEF, 4E-BP1 phosphorylation was low under conditions of serum
starvation but was strongly induced by PDGF within 15 min. Like S6K,
4E-BP1 was constitutively phosphorylated in P3k- or Akt-transformed
CEF, even when the cells were serum deprived. For comparison,
phosphorylation of Erk was low in serum-starved P3k- or Akt-transformed
cells and was efficiently induced to the level seen in serum-starved
CEF stimulated with PDGF.

Constitutive phosphorylation of S6K and 4E-BP1 in CEF transformed with
P3k and Akt. CEF and CEF infected with RCAS, RCAS-Myr-Akt, and
RCAS-Myr-c-P3k were serum starved for 40 h and then stimulated
with PDGF for 15 min. The cells were lysed, and the lysates were
resolved in a 10% (A) or a 15% (B)
SDS-polyacrylamide gel and then transferred to a polyvinylidene
difluoride membrane. The blot was probed with anti-phospho-S6K
(threonine 389), anti-S6K, anti-phospho-4E-BP1 (serine 65),
anti-4E-BP1, or anti-phospho-Erk antibody.

The Oncogenic Activity of Akt Is Correlated with the Ability to
Induce Phosphorylation of S6K and of 4E-BP1.

The oncogenicity of Akt requires both membrane localization and
specific enzymatic activity (12). Myristylated Akt is highly oncogenic,
whereas the construct with the regulatory phosphorylation sites
threonine 308 and serine 473 mutated to aspartic acid induces cellular
transformation only with a very low efficiency, despite the fact that
this mutant retains high kinase activity (12). Western blots of CEF
infected with the Akt-DD mutant showed no elevated phosphorylation of
S6K, in contrast to Myr-Akt-transformed CEF (Fig.
2A). Similarly,
phosphorylation of 4E-BP1 was constitutively increased in
Myr–Akt-infected cells but not in Akt–DD-infected cells (Fig.
2B). These results suggest a correlation between
oncogenicity and the phosphorylation of S6K and of 4E-BP1. [Thomas and
collaborators recently reported that Akt-DD could phosphorylate 4E-BP1
but not S6K (54). The discrepancy with our observations may be due to
the mode of Akt-DD expression—transient transfection versus stable
transfection—or may reflect the use of different cell types. It is
also possible that 4E-BP1 is partially phosphorylated by Akt-DD but not
fully inactivated.]

Correlation between the ability to induce S6k activation and 4E-BP1
inactivation with the ability to induce oncogenic transformation. Cell
lysates were prepared as described in the legend to Fig. 1. The lysates
were resolved in a 10% (A) or a 15% (B)
SDS-polyacrylamide gel. The blot was probed with anti-phospho-S6K
(threonine 389), anti-S6K, or anti-phospho-4E-BP1 (serine 65)
antibody.

Rapamycin Inhibits Formation of Transformed Cell Foci by P3K in an
FKBP12-Dependent Manner.

The phosphorylation of S6K is known to be rapamycin-sensitive (55, 56).
Since phosphorylation is correlated with oncogenic transformation by
P3k and Akt, the target of rapamycin, mTOR, might be involved in
cellular transformation induced by P3k or Akt. This possibility was
explored by determining the effect of rapamycin on the transforming
efficiency of P3k. As shown in Fig. 3, 1
ng/ml rapamycin almost completely eliminated the formation of
transformed cell foci by P3k. This inhibition was specific because even
20 ng/ml rapamycin did not significantly influence focus formation by
the oncoproteins Src or Jun, although it induced a slight delay in
transformation, possibly due to partial growth inhibition caused by the
drug in CEF. Rapamycin inhibits mTOR by binding to the immunophilin
FKBP12 (54). The interference with P3k transformation probably also
involves interaction with FKBP12 because a 100-fold molar excess of
FK506, a pharmacological reagent that competes with rapamycin for
binding to FKBP12, rescued P3k transformation (Fig.
4).

Rapamycin inhibits focus formation by P3k but not Jun or Src. CEF were
infected with viruses containing the indicated oncoproteins. Each plate
was infected with 100 μl of the virus stocks diluted to
10−1 (top left well), 10−2 (top center well),
10−3 (top right well), 10−4 (bottom left
well), 10−5 (bottom center well), or with no viruses
(bottom right well). The cells were overlaid with nutrient agar
containing the indicated concentrations of rapamycin (RAPA) for 17 days
and then fixed and stained with crystal violet.

The Inhibitory Effect of Rapamycin on Oncogenic Transformation Is
Specific for P3k and Akt.

The acute sensitivity of P3k-induced oncogenic transformation to
inhibition by rapamycin, compared with the refractoriness of Jun and
Src, prompted us to test the effect of rapamycin on focus formation
caused by other oncoproteins. Akt was the only other oncoprotein that
showed sensitivity to rapamycin, reflecting the fact that the Akt
protein is part of the PI 3-kinase signaling pathway and suggesting
that rapamycin intervenes downstream of Akt. Besides the
above-mentioned Src and Jun, the oncoproteins Abl, Crk, ErbB, Fos, Fps,
Mos, Qin, Sea, and Yes were not significantly affected in their
transforming activity by rapamycin. Surprisingly, the growth-promoting
potential of two oncoproteins, Myc and Ras, was strongly enhanced by
rapamycin, leading to an increase in the number and size of the
transformed cell foci (Table 1).

Effects of rapamycin on transforming activities of various
oncogenes in CEF

Rapamycin Inhibits the Constitutive Phosphorylation of S6K in CEF
Transformed by P3k or Akt.

The suggested link between the activating phosphorylation of S6K and
oncogenic transformation was strengthened by examining S6K
phosphorylation in the presence and absence of rapamycin (Fig.
5). Rapamycin led to an almost complete
disappearance of the phosphorylated form of S6K, not only from normal
CEF but also from CEF transformed by P3k or Akt. Although
transformation by P3k or Akt induces a constitutive phosphorylation of
S6K, this activation of S6K is still rapamycin-sensitive as is
transformation itself.

Rapamycin inhibits phosphorylation of S6K. CEF were serum starved for
40 h and treated or not treated with rapamycin (RAPA, 10 ng/ml)
for 2 h and then stimulated with PDGF (50 ng/ml) for 15 min. The
cell lysates were prepared and resolved in a 10% SDS-polyacrylamide
gel. The blot was probed with anti-phospho-S6K or with anti-S6K
antibody.

Discussion

The kinase mTOR is at the center of the experiments reported here.
mTOR is inhibited by the macrolide antibiotic rapamycin; rapamycin
interacts with FKBP12, and this complex binds to mTOR (57–60). FK506
competes with rapamycin in binding to FKBP12 and thus counteracts the
inhibition of mTOR (61, 62). Rapamycin is effective at very low
concentrations in eliminating mTOR kinase activity (57–60). Since the
inhibition of mTOR also abolishes the oncogenic effects of P3k and of
Akt, mTOR appears to be an obligatory mediator of the oncogenic signal
issued by P3k or Akt. mTOR is phosphorylated, probably directly, by Akt
at two carboxyl-terminal sites (63). These two sites are located in a
negative regulatory domain of mTOR; phosphorylation may relieve the
negative regulation and activate the mTOR kinase (63). Although the
complete target spectrum of mTOR remains to be determined, it is clear
that mTOR functions as an important regulator of translation. mTOR
mediates the phosphorylation of S6K and of 4E-BP1 (47–52, 64). Active
S6K is essential for the translation of 5′-TOP mRNAs, which include
messages of ribosomal proteins (28). Inactivation of 4E-BP1 facilitates
the translation of mRNAs with complex 5′ secondary structures; many of
these code for growth-related genes (26, 65).

Our observations suggest that some mTOR-dependent activity, possibly
connected to S6K and 4E-BP (and hence linked to eIF4E), is essential
for oncogenic transformation induced by P3k or Akt. Transformation by
11 diverse oncoproteins was refractory to inhibition by rapamycin, and
transformation by two, Ras and Myc, was enhanced. Some of the
rapamycin-refractory oncoproteins, for instance the Src kinase and the
adaptor protein Crk, are known to activate PI 3-kinase–Akt signaling
(66). Src also induces phosphorylation of S6K (M.A. and P.V.,
unpublished observations). The resistance to rapamycin indicates that
the branch of the PI 3-kinase–Akt signal that is transduced by mTOR is
not irreplaceable or essential in the oncogenicity of Src or Crk. In
the case of Src, oncogenic signals appear to proceed through two
alternative routes, involving mTOR on the one hand and the
Ras–mitogen-activated protein kinase pathway on the other. Both must
be inhibited to interfere with cellular transformation (67). These
comparisons of rapamycin sensitivity and resistance single out P3k and
Akt as following a unique mechanism of transformation, not commonly
shared by other oncoproteins. A recent publication reported that
transformation of an immortalized rat kidney epithelial cell line by
the zinc finger transcription factor GLI is also rapamycin sensitive,
and this sensitivity is correlated with an inhibition of protein
synthesis. Transformation of these same cells by Ras or Myc is not
inhibited by the antibiotic (68). The available data support the
general statement that translational controls are an essential
component of oncogenic transformation by P3k, Akt, or GLI.

Previous studies have suggested an oncogenic potential for
several regulators of protein synthesis (for review, see ref. 69).
eIF4E was shown to transform NIH 3T3 cells (70, 71), and mutated S6K
can affect the morphology of Rat1 cells (72). A cautious interpretation
of these data suggests that a gain of function in S6K or of eIF4E may
be necessary for certain mechanisms of transformation but is possibly
not sufficient. Besides eIF4E, there are two other eukaryotic
initiation factors that have shown oncogenic potential, eIF-2A and
eIF-4G (73, 74). 4E-BP1 is also hyperphosphorylated and thus
inactivated in Src-transformed hamster fibroblasts (75).

There is evidence that PI 3-kinase and Akt act as oncogenic
determinants in several human cancers. PIK3CA, the catalytic subunit of
class IA PI 3-kinase, is overexpressed in ovarian cancers (76). Loss of
function in PTEN, a phosphatase that counteracts PI 3-kinase, is a
frequent genetic change in numerous tumors including carcinoma of the
prostate and glioblastoma (77–79). mTOR is constitutively
phosphorylated in prostate cancer cell lines carrying an inactivating
mutation in PTEN or overexpressing Akt3 (63). Akt genes are amplified
or overexpressed in several cancers including gastric, ovarian, breast,
pancreatic, and prostate cancer (80–85); eIF4E is overexpressed in
lymphomas, cancers of the head and neck and in colon carcinomas as well
as in cells containing elevated levels of the oncoprotein Myc (86). An
importance of S6K in human cancer is suggested by the frequent
up-regulation of mRNAs for ribosomal proteins in expression profiles
from diverse tumors (87). Several cell lines derived from human tumors,
including rhabdomyosarcomas and small cell lung carcinomas, are
sensitive to rapamycin (88, 89).

The special mechanisms of transformation suggested here for P3k and Akt
may therefore be relevant to human cancer. Akt is an obvious target for
the design of novel chemotherapeutic agents, but it might not be
desirable to inhibit all of the multiple functions of Akt. mTOR and its
subordinate activities may provide opportunities for a chemotherapeutic
strategy that is more narrowly aimed and more selective.

Acknowledgments

We thank K. C. Nicolaou, Daniel R Salomon, and Klaus Hahn for
valuable comments on the manuscript. Osvaldo Batista and Jeffrey Ludwig
provided competent and dedicated technical assistance. This work was
supported by National Institutes of Health Research Grants CA42564,
CA78230, and CA79616. This is manuscript number 13607 of the Department
of Molecular and Experimental Medicine, The Scripps Research Institute.

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